NEC is the most serious gastrointestinal disorder of premature infants and one of the leading causes of death in neonatal intensive care units (NICU). It is the most common surgical emergency in the newborn period and the second leading cause of morbidity and mortality in the preterm population. The incidence of NEC in selected studies has ranged from fewer than 1% to as many as 5% of NICU admissions (Stoll B J: Epidemiology of necrotizing enterocolitis. Clin Perinatol 21:205-218, 1994.)
A recent multicenter study of 2681 infants weighing 501-1500 grams reported that proven NEC (Bell Stage 2-3) occurred in 10.1% and suspected NEC (Bell Stage 1) in a further 17.2% of the cohort; mortality was 54% in infants with severe (Stage 3) NEC. These data indicate that NEC is a major public health problem in neonates: given the .about.4 million births/year in the United States, NEC would be expected to develop in 1200-9600 infants, of whom between 9-28% will die as a result of their disease. (Walsh M C, Kleigman R M, Fanaroff A A: Necrotizing enterocolitis: A practitioner's perspective. Pediatr Rev 9:219-226, 1988; Uauy R D, Fanaroff A A, Korones S B, Phillips E A, Philips J B, Wright L: Necrotizing enterocolitis in very low birth weight infants: Biodemographic and clinical correlates. J Pediatr 119:630-38, 1991.)
Earlier studies indicated a mortality of 10-55% in premature infants (Stoll B J, Kanto W P, Glass R I, Nahmias A J, Brann A W: Epidemiology of necrotizing enterocolitis: A case control study. J Pediatr 96:447-451, 1980).
Survivors of NEC can also have considerable long-term morbidity resulting from their disease, including short-gut syndrome, failure-to-thrive, intestinal stricture, and the need for repeated surgery (Simon N P: Follow-up for infants with necrotizing enterocolitis. Clin Perinatol 21:411-424, 1994).
NEC is primarily a disease of premature infants who have survived the immediate neonatal period. There is an increased incidence of NEC with decreasing birthweight and gestational age; in a population-based study involving all infants born in a single state during a 1-year period, the incidence of NEC was 42.1/1000 livebirths weighing &lt;1000 grams, compared with only 3.8/1000 in the 1500-2501 gm birthweight category, and 0.11/1000 in children weighing &gt;2500 grams at birth (Wilson R, Canto W P, McCarthy B J et al.: Epidemiologic characteristics of necrotizing enterocolitis: a population based study. Am J Epidemiol 114:880-887, 1981.)
The recent introduction of replacement surfactant therapy has resulted in a decrease in the mortality rate associated with respiratory distress syndrome in extremely low-birthweight infants, precisely that population at highest risk for developing NEC. With the prevalence of NEC likely to rise, NEC may become an increasingly important cause of neonatal mortality in the 1990's.
All premature infants are at risk of developing NEC, with the risk increasing with decreasing gestational age. Of 4 million live births/yr in the U.S., approximately 8% (320,000) are prematurely born. All of these infants will be eligible to receive multiple doses of Lactobacillus/Bifidobacteria preparation. Currently the clinical management of NEC is largely empiric consisting of general supportive measures. Cost of the intensive medical care per patient with NEC ranges between $50,000-$100,000; with even higher hospital cost for those requiring surgery. In spite of all these interventions, mortality rate in NEC has been reported to be upto 55%.
In addition, those undergoing surgery are often left with long term dysfunctions such as short gut syndrome and failure to thrive, translating to continued morbidity and medical expense.
Stool Microfloral Colonization Patterns in the Newborn Period
Bacterial colonization of the neonatal gastrointestinal tract begins when maternal cervical and vaginal flora is encountered by the infant during delivery. (Brooke I, Barrett C T, Brinkman C R, Martin W J, Finegold S M: Aerobic and anaerobic bacterial flora of the maternal cervix and newborn gastric fluid and conjunctiva: A prospective study. Pediatrics 63:451-455, 1979).
By 10 days of age the large majority of healthy full-term newborns are fully colonized with a variety of bacterial species (Long S S, Swenson R M: Development of anaerobic fecal flora in healthy newborn infants. J Pediatr 91:298-301, 1977.)
In contrast, the gut of a premature infant, cared for in relatively aseptic NICU conditions and usually receiving antibiotics shortly after birth, does not get the opportunity for proper colonization by the normally heterogeneous bacterial flora and demonstrates delayed colonization with a limited number of bacterial species (Gupta S, Morris J G, Panigrahi P, Natero J P, Glass R I, Gewolb I H: Endemic necrotizing enterocolitis:lack of association with a specific infectious agent. Pediatr Infect Dis 13:725-734, 1994; Goldman D A, Leclair J, Macone A: Bacterial colonization of neonates admitted to an intensive care environment. J Pediatr 93: 288-93, 1978; Bennet R, Eriksson M, Nord C E, Zetterstrom R: Fecal bacterial microflora of newborn infants during intensive care management and treatment with five antibiotic regimens. Pediatr Infect Dis J 5:533-539, 1986).
Indeed, we have recently shown that the stool of preterm infants, with and without NEC, is colonized on the average by fewer than 2.5 species of aerobic bacteria, compared to &gt;10 species in fullterms (Gupta S, Morris J G, Panigrahi P, Natero J P, Glass R I, Gewolb I H: Endemic necrotizing enterocolitis:lack of association with a specific infectious agent. Pediatr Infect Dis 13:725-734, 1994.)
Human milk populates the intestine with Bifidobacteria and Lactobacilli, generating a very different gut flora than that seen after formula feeding (Keyworth N, Miller M R, Holland K T: Development of cutaneous microflora in premature Neonates. Arch Dis Child 67:797, 1992; Yoshioka H, Iseki K, Fujita K: Development and differences of intestinal flora in the neonatal period in breast-fed and bottle-fed infants. Pediatrics 73:317-321, 1983.) It has long been known that Bifidobacteria are the most common organisms found in newborn fecal material. Yoshioka et al. used quantitative culture methods to study differences in intestinal colonization patterns in breast- and bottle-fed full term neonates. (Yoshioka H, Iseki K, Fujita K: Development and differences of intestinal flora in the neonatal period in breast-fed and bottle-fed infants. Pediatrics 73:317-321, 1983).
Both groups were first colonized with Enterobacteria. However, by day 6, Bifidobacteria exceeded Enterobacteria by 1000:1 in breast-fed infants; in bottle-fed infants, Enterobacteria outnumbered Bifidobacteria by 10:1. By one month of age, Bifidobacteria was the predominant species in both groups, but the absolute number of these organisms was 10 times fewer in bottle-fed babies compared to the breast-fed group.
Of interest, a recent study demonstrated a higher incidence of aerobic flora in preterm NICU infants fed frozen human milk, as well as an increased rate of isolation of S. epidermidis. (El-Mohandes A, Keiser J F, Johnson L A, Refat M, Jackson B J: Aerobes isolated in fecal microflora of infants in the intensive care nursery: Relationship to human milk use and systemic sepsis. Am J Infect Control 21:231-234, 1993.)
A number of investigators have found decreased numbers of Lactobacilli in preterm infants; this reduction was correlated with previous antibiotic therapy and time spent in an incubator (Hall M A, Cole C B, Smith S L, Fuller R, Rolles C J: Factors influencing the presence of faecal lactobacilli in early infancy. Arch Dis Child 65:185-188, 1990.)
Delayed transit time, seen in preterm infants could permit bacterial overgrowth that could, in turn, initiate the cascade of events that lead to NEC. This finding again underscores the potential for overgrowth by a small number of potential pathogens in the low birth weight infant in the NICU. (Vantrappen G, Janssens J, Choos Y: The interdigestive motor complex of normal subjects and patients with bacterial overgrowth of the small intestine. J Clin Invest 59:1158, 1977).
The experience with germ-free animals may be applicable to the situation existing in the preterm infant. If germ free animals are contaminated with a single strain of organism, that organism, even if not normally indigenous or pathogenic, can populate the gut in very high concentrations (Gibbons R J, Socransky S S, Kapsimalis B: Establishment of human indigenous bacteria in germ free mice. J Bacteriol 88:1316-1323, 1964; Schaedler R W, Dubos R, Costello R: Association of germ free mice with bacteria isolated from normal mice. J Exp Med 122:77-82, 1965.)
Germ free pigs and guinea-pigs can develop severe enteritis when removed from the germ-free environment or when contaminated with a single species (Tanami J: J. Chiba Med. Soc. 35:1, 1959; quoted in Luckey T D: Effects of microbes on germ free animals. Adv Appl Microbiol 7:169-223, 1965); (Moberg L J, Sugiyama H: Microbial ecological basis of infant botulism as studied with germfree mice. Infect Immunol 25:653-657, 1979); of note, protection can be afforded by prior seeding with elements of their normal stool flora (Tanami J: J. Chiba Med. Soc. 35:1, 1959; quoted in Luckey T D: Effects of microbes on germ free animals. Adv Appl Microbiol 7:169-223, 1965; Moberg L J, Sugiyama H: Microbial ecological basis of infant botulism as studied with germfree mice. Infect Immunol 25:653-657, 1979.)
"Schaedler's Cocktail," a combination of harmless bacteria, is routinely used in raising specific pathogen-free rodents; without such treatment, the normal physiologic development of the gut does not occur, and the rodents become susceptible to infection with bacteria that would otherwise be considered normal flora for the particular strain of rodent (Harp J A, Chen W, Harmsen A G: Resistance of severe combined immunodeficient mice to infection with Cryptosporidium parvum: the importance of intestinal microflora. Infect Immun 60:3509-3512, 1992.)
Stool Microbial Patterns in NEC
There have been a number of studies of the enteric flora in infants with NEC. Blakely et al. found a decreased percentage of infants with NEC harboring Bacteroides spp. (32% vs 61% in controls) and Lactobacilli (12% vs 48% in controls), and an increased number with C. perfringens (40% vs 13%). (Blakely J L, Lubitz L, Campbell N T, Gillan G L, Bishop R F, Barnes G L: Enteric colonization in sporadic neonatal necrotizing enterocolitis. J Pediatr Gastroenterol Nutr 4:591-595, 1985).
Bell et al. noted increased numbers of Gram (-) bacteria (especially E. Coli and Klebsiella spp.) in infants with NEC. (Bell M J, Feigin R D, Ternberg J L, Brotherton T: Evaluation of gastrointestinal microflora in necrotizing enterocolitis. J Pediatr 92:589-91, 1978).
In contrast, in a prospective case-control study of endemic NEC, we were unable to demonstrate any significant associations between any single aerobic bacterial species and NEC (Gupta S, Morris J G, Panigrahi P, Natero J P, Glass R I, Gewolb I H: Endemic necrotizing enterocolitis:lack of association with a specific infectious agent. Pediatr Infect Dis 13:725-734, 1994).
Lactobacillus/Bifidobacterial Therapy
Fuller has described the utility of live microbial feed supplement which can beneficially affect the host animal by improving its microbial balance (Fuller R: Probiotics in man and animals. J Applied Bacteriol 66:365-378, 1989; Fuller R: Probiotics in human medicine. Gut 32:439-442, 1991.)
There are a number of lines of evidence pointing to the potential benefits of such a therapy (Fuller R: Probiotics in human medicine. Gut 32:439-442, 1991.)
Our studies on the gut ecology of preterm infants with and without NEC, has demonstrated with compelling evidence that only certain Gram (+) organisms that are capable of abrogating the adherence of Gram (-) bacteria can be utilized to block NEC-like disease in rabbit ileal loops (Panigrahi P, Gupta S, Gewolb I H, Morris J G: Occurrence of necrotizing enterocolitis may be dependent on patterns of bacterial adherence and intestinal colonization: Studies in Caco-2 tissue culture and weanling rabbit models. Pediatr Res 36:115-121, 1994.)
A variety of in vitro studies now indicate that intestinal bacteria can inhibit pathogenic bacteria. Sullivan et al. showed that gut isolates of Bifidobacteria, Lactobacilli, Propionibacteria, and Enterococci inhibit C. botulinum in vitro. (Sullivan N M, Mills D C, Riemann H P, Arnon S S: Inhibitions of growth of C. botulinum by intestinal microflora isolated from healthy infants. Microbial Ecology in Health and Disease 1:179-192, 1988). Numerous in vivo studies also lend support to the ability of selected Lactobacilli to modify the intestinal microflora (Conway P: Lactobacilli: Fact and fiction. Ch. 16 in The regulatory and protective role of the normal flora. Grun R, Midvedt T, Norin E, eds. 1988. Stockton Press, pp. 263-281).
For example, seeding the gut with Lactobacilli significantly reduces the numbers of E. Coli in chickens, pigs, and humans. (Fuller R: Epithelial attachment and other factors controlling the colonization of the intestine of the gnotobiotic chicken by lactobacilli. J Applied Bacteriol 45:389-395, 1978; Barrow P A, Brooker B E, Fuller R, Newport M J: The attachment of bacteria to the gastric epithelium of the pig and its importance in the microbiology of the intestine. J Applied Bacteriol 48:147-154, 1980; Lidbeck A, Gustafsson J-A, Nord, C E: Impact of Lactobacillus acidophilus supplements on the human oropharyngeal and intestinal microflora. Scand J Infect Dis 19:531-537, 1987).
Using the rabbit ileal loop model, both, Foster et al. and Johnson and Calia, showed that Lactinex (a commercial preparation containing L. acidophilus and L. bulgaricus) decreased loop fluid production caused by enterotoxigenic E. Coli. (Foster T L, Winans L, Carski T R: Evaluation of lactobacillus preparation in enterotoxigenic E. Coli-induced rabbit ileal loop reactions. Am J Gastroenterol 73:238-243, 1980; Johnson D E, Calia F M: The effect of Lactinex on rabbit ileal loop reactions induced by enterotoxigenic Escherichia coli. Curr Microbiol 2:207-210, 1979).
An adherent strain of Bifidobacterium bifidum has also been used to mediate the clinical course of rotavirus diarrhea in mice (Duffy L C, Zielezny M A, Riepenhoff-Talty M, Dryja D, Sayahtaheri-Altaie S, Griffiths E, Ruffin D, Barrett H, Rossman J, Ogra P L: Effectiveness of Bifidobacterium bifidum in mediating the clinical course of murine rotavirus diarrhea. Pediatr Res 35:690-695.)
Although these preparations have been used all over the world for decades, more so in European, Scandinavian, and Asian countries, their use has been more empiric than with specific scientific rationale. In turn, large and sometimes contradictory results have been reported on the use of Lactobacilli to treat diarrhea in older children and adults. The selection of Lactobacillus/Bifidobacteria strains have been random without any understanding of their inherent characteristics, and without concurrent consideration of the pathogenesis of the disease in which it is being used.
Effect of Lactobacillus/Bifidobacteria on Neonatal Gut Flora
There have been a limited number of attempts to modify the gastrointestinal flora in the neonatal period. In a double-blind clinical trial with 30 preterm infants (mean weight 1350 gm), Reuman et al. studied the effect of introducing 12 hr oral feedings of 10.sup.9 Lactobacillus acidophilus within 72 hours of delivery. Stool was cultured weekly for Lactobacilli, for Gram (-) bacteria, and for antibiotic resistance of the Gram (-) bacteria. (Reuman P D, Duckworth D H, Smith K L, Kagan R, Ayoub E M: Lack of effect of Lactobacillus on gastrointestinal bacterial colonization in premature infants. Pediatr Infect Dis 5:663-668, 1986).
Lactobacilli were recovered in the stools of 13/15 infants receiving the Lactobacillus supplementation vs 3/15 of the control group. Lactobacilli were also recovered significantly earlier from the treated group (at 19 vs 47 days). No difference was found in the isolation rate of Gram (-) bacteria or in the number of antibiotic-resistant organisms. The details of specific types of Gram (-) organisms were not given, nor was there an attempt to do quantitative cultures.
The choice of Lactobacillus acidophilus was arbitrary, and not based on adherence or other characteristics that might determine ability to successfully colonize the intestine (Barrow P A, Brooker B E, Fuller R, Newport M J: The attachment of bacteria to the gastric epithelium of the pig and its importance in the microbiology of the intestine. J Applied Bacteriol 48:147-154, 1980).
However, Mautone et al. have demonstrated a reduction in Gram (-) flora and a percentage increase in Gram (+) bacilli after feeding a suspension containing Bifidobacterium bifidum and Lactobacillus acidophilus to a group of 77 neonates (61 fullterm; 16 preterm). More recently, Bennett et al. showed that orally administered Bifidobacteria and Lactobacilli could be cultured from the feces of newborn fullterm infants after antibiotic treatment for a number of days after such therapy was discontinued. (Lidbeck A, Gustafsson J-A, Nord, C E: Impact of Lactobacillus acidophilus supplements on the human oropharyngeal and intestinal microflora. Scand J Infect Dis 19:531-537, 1987; Bennet R, Nord C E, Zetterstrom R: Transient colonization of the gut of newborn infants by orally administered bifidobacteria and lactobacilli. Acta Paediatr 81:784-787, 1992).
In conclusion, these and other studies indicate that it is possible to successfully modify the gut flora in preterm infants by orally administered Bifidobacteria and Lactobacilli during and after antibiotic therapy. It is almost impossible to avoid using antibiotics in most preterms receiving treatment for other ailments.
We have demonstrated that adherent Gram (-) bacteria are capable of causing NEC-like disease in animal models. We have also shown that it is not the species or strain of bacteria that is responsible for evoking an injury response, rather the microbial ecology (combination of Gram (-) and Gram (+) bacteria) of the premature gut that either protects or gives rise to a cascade of events ultimately resulting in NEC. (Gupta S, Morris J G, Panigrahi P, Natero J P, Glass R I, Gewolb I H: Endemic necrotizing enterocolitis:lack of association with a specific infectious agent. Pediatr Infect Dis 13:725-734, 1994; Panigrahi P, Gupta S, Gewolb I H, Morris J G: Occurrence of necrotizing enterocolitis may be dependent on patterns of bacterial adherence and intestinal colonization: Studies in Caco-2 tissue culture and weanling rabbit models. Pediatr Res 36:115-121, 1994).
The selection of Lactobacillus/Bifidobacteria strains have been random without any understanding of their inherent characteristics, and without concurrent consideration of the pathogenesis of the disease in which it is being used. There is a need in the art for the development of an appropriate strain specifically for premature infants (not full term). The invention described below fulfills this need by providing two specific strains of Lactobacillus which clearly demonstrate reduced tissue injury and reduced acute inflammatory cell infiltration. The present invention is useful in prevention and/or treatment of NEC in premature infants.